Rapid changes in the rhizosphere bacterial community structure during re-colonization of sterilized soil

Diversity has been shown to be pivotal in ecosystem stability and resilience. It is therefore important to increase our knowledge about the development of diversity. The aim of this study was to investigate the temporal dynamics of the bacterial community structure in the rhizosphere of wheat plants growing in a soil in which the initial conditions for bacterial re-colonization were modified by mixing different amounts of sterilized with native soil at ratios of 19:1, 9:1, 4:1 and 1:1. Additional treatments comprised sterilized soil or native soil. Plant dry weight at day 20 decreased with increasing percentage of native soil in the mix. The bacterial community structure in the rhizosphere was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) at days 3, 14 and 20 after planting. The bacterial community in the sterilized soil had a lower diversity and evenness than the native soil. Both diversity and evenness increased with time in the sterilized soil. Community structure in the different mixes changed over time and the changes were mix-specific. Principal component analyses of the DGGE banding patterns showed clear differences between the treatments particularly at day 3 and day 14 and revealed changes in community structure within a few days in a given treatment. The results of the present study show that bacterial communities rapidly re-colonize sterilized soil. During re-colonization, the community structure changes rapidly with a general trend towards higher diversity and evenness. The changes in community structure over time are also affected by the amount of sterile substrate to be re-colonized.     

断奶前后仔猪十二指肠、空肠和回肠SGLT1 mRNA表达的变化及半胱胺的影响

研究断奶前后仔猪十二指肠、空肠和回肠SGLT1mRNA表达丰度的变化,并同时观察半胱胺对其表达的影响,从分子水平探讨半胱胺对断奶仔猪葡萄糖吸收的影响与可能的机制。新生仔猪随机分为对照组和实验组,12日龄(D12)起对照组饲喂基础乳猪料,实验组在基础乳猪料中添加120mg/kg半胱胺,两组仔猪均于35日龄断奶,每组分别于断奶前1周、断奶当天、断奶后3d、1周以及断奶后10d随机选取仔猪各6头,宰杀后采集十二指肠、空肠和回肠粘膜,采用RT-PCR方法测定钠-葡萄糖共转运载体1(sodium-glucosecotransporter1,SGLT1)mRNA表达的相对丰度。结果表明,回肠SGLT1mRNA的表达量最高,空肠其次,十二指肠最低;SGLT1mRNA在不同肠段的表达呈现截然不同的发育模式,十二指肠SGLT1mRNA表达量在45d显著上升(P<0.05),而空肠则在42和45d时显著下降(P<0.01),回肠SGLT1mRNA表达在整个实验期间无显著变化;此外,日粮添加半胱胺显著提高38d十二指肠以及42和45d空肠SGLT1mRNA表达,而对回肠SGLT1mRNA表达无显著影响。小肠SGLT1mRNA表达以及日粮添加半胱胺促进SGLT1mRNA表达的作用均呈现时空特异性规律。     

PCR-DGGE技术用于猪场沼气池细菌群落分析的条件优化

本研究采用变性梯度凝胶电泳（PCR-DGGE）技术研究猪场沼气池细菌群落,对基于16S rDNAV3区的PCR-DGGE电泳的最佳变性剂梯度范围、电泳时间、染色时间进行优化。研究结果表明,最佳变性剂梯度范围为35%～60%,电泳时间为12h,SYBR Green Fluorescent Dye染料的染色时间是30min,优化后的PCR-DGGE确保了实验的准确性、灵敏度和可重复性。运用此优化后的PCR-DGGE技术对5个猪场沼液细菌群落进行了研究,获得了较丰富的多样性。该实验为深入研究畜禽养殖粪污治理的菌种筛选奠定了基础。     

Effect of organic mulches on soil bacterial communities one year after application

The application of organic mulches as a soil cover is effective in improving the quality of soil. However, very little information is available on the effect of mulches on the soil microbial community. In this study, we investigated the effect of various organic mulches on soil dehydrogenase activity (DHA) and microbial community structures in the top 1 cm and 5 cm below the soil surface 1 year after application of the mulches. DHA was stimulated at both depths in plots mulched with grass clippings (GC), but was not significantly different from the control for the other mulch treatments. Fatty acid methyl ester (FAME) analysis and denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rDNA fragments were used to assess changes in the soil microbial community structure. Cluster analysis and principle component analysis of FAME profiles showed that only soil mulched with pine chips distinctively clustered from the other treatments. At the soil surface, bacterial DGGE profiles revealed that distinct shifts in several bacterial populations occurred in soils mulched with GC and eucalyptus yardwaste (EY), while DGGE profiles from soil at the 5 cm depth revealed no distinct changes. Changes in bacterial diversity at the soil surface under different mulches were calculated based on the number of bands in the DGGE profile using the Shannon-Weaver index of diversity ( H). Compared to the control ( H =0.9), the GC- and EY-treated soils showed slightly increased bacterial diversity, with an H of 1.1 and 1.0, respectively. These results indicate that the long-term effect of organic mulches on the soil microbial activity and community structure is highly dependent upon the type of mulch and is mostly exerted in the top few centimeters of the soil profile.     

小肽营养素对断奶仔猪生产性能及血液生化指标的影响

本试验将40头21d断奶的三元杂交猪随机分为4组,用4组不同日粮组成及营养水平的饲料（Ⅰ：基础日粮＋2%喷雾干燥血浆蛋白粉（SDPP）;Ⅱ、Ⅲ和Ⅳ组分别在基础日粮中添加1%,2%和3%小肽营养素）饲养21d后称重、测定血液生化指标并进行统计分析。结果表明：添加小肽营养素对断奶仔猪的生产性能无显著影响;各处理组的血清总蛋白、血糖和血清尿素差异不显著,2%小肽组的球蛋白、免疫球蛋白含量显著高于其它组;小肽产品可以部分等蛋白取代断奶仔猪日粮中的血浆蛋白粉。通过了解小肽营养素替代喷雾干燥血浆蛋白粉对21d断奶仔猪的生产性能和血液生化指标的影响,为肽类产品在断奶仔猪日粮中的应用提供了依据。     

鸡粪好氧堆肥过程中氨氧化古菌群落结构的动态变化

应用聚合酶链式反应-变性梯度凝胶电泳（PCR DGGE）技术,研究了好氧堆肥过程氨氧化古菌（ammonia oxidizing archaea, AOA）的群落结构和多样性变化。结果表明,不同堆肥时期鸡粪好氧堆肥AOA菌群的群落结构发生了明显的变化。与AOA HH 2(GU2258721)亲缘关系较近的b条带(相似性96%)和未培养泉古菌属［uncultured crenarchaeote NM 152(HQ8752251)］的m条带(相似性99%)是堆肥过程一直存在的AOA菌属。条带c、 b、 f、 i和条带m、 k、 l、 n代表的种属分属两个大类。AOA群落的Shannon Weiner指数（H）、 均匀度指数（EH）均表现为第30 d＞第5 d＞第25 d第45 d＞第3 d第12 d＞第1 d第15 d。对AOA种群数据与不同时期堆体温度、 pH、 全氮、 铵态氮、 亚硝态氮和硝态氮等环境因子的冗余分析表明,堆体温度、 全氮、 亚硝态氮和硝态氮对氨氧化古菌群落演替有着显著的影响（P0.05）,AOA的群落结构在堆肥第1 d、 第5 d、 第25 d、 第30 d、 第45 d变化较大。说明这些参数能有效控制鸡粪好氧堆肥过程中AOA的群落结构。     

生草栽培对南丰蜜橘园土壤微生物群落结构与功能特征的影响

为探究橘园生草对土壤微生物群落结构与功能特征的影响,本研究以南丰蜜橘(Citrus reticulita Blanco cv.Kinokuni)为研究对象,设置行间播种白三叶草(Trifolium repens)、黑麦草(Lolium multiflorum)、自然生草和清耕4个处理,采用荧光定量PCR技术和Illum...     

不同栽培环境下有机与常规蔬菜土壤的细菌群落多样性差异

土壤细菌群落在蔬菜栽培中发挥着重要作用。基于DNA和RNA水平,利用PCR-DGGE技术研究了不同栽培环境下有机与常规蔬菜土壤细菌群落多样性差异,以及土壤理化性质与细菌群落多样性的关系。结果表明：不同栽培方式下土壤细菌多样性存在明显差异,土壤微生物的优势种群和数量受有机、常规栽培和季节影响,有机栽培较之常规栽培能够显著增加土壤细菌群落多样性;聚类分析表明,16S rDNA细菌群落多样性与季节相关,而16S rRNA细菌群落多样性与栽培方式相关;差异条带测序显示,大多细菌与不可培养细菌种属有较高同源性,其余9种推测属于假单胞菌属;CCA分析说明pH是影响土壤细菌群落多样性的主要因素,有机栽培土壤中微生物生物量C、N以及有机质含量显著高于常规栽培土壤。综上,有机栽培能够丰富活性细菌群落多样性,具有土壤优化效应。     

The impact of increasing heavy metal stress on the diversity and structure of the bacterial and actinobacterial communities of metallophytic grassland soil

A study was undertaken to investigate the bacterial community found in metallophytic grassland soil contaminated with Zn and Pb. We hypothesised that such communities would be tolerant of additional heavy metal stress due to phylogenetic and functional adaptation. In microcosm experiments, lasting 51 days, denaturing gradient gel electrophoresis (DGGE) analyses was used to compare the total bacterial and actinobacterial communities in non-amended soils and those to which additional Pb and Zn concentrations were added. There was a decrease in total bacterial diversity with each addition of Pb and Zn; in contrast, the actinobacterial community diversity remained unaffected. The community structures were analysed using multivariate analyses of the DGGE profiles. Total bacterial community profiles showed two distinct groups sharing less than 80% similarity, irrespective of Pb and Zn addition. The first contained profiles sampled during the first 7 days of the experiment; the second contained those sampled from day 10 onwards. Actinobacterial profiles from those that were non-amended showed a similar distribution to those of the total bacterial community. However, in soil amended with fivefold additional Pb and Zn, all the profiles shared more than 80% similarity. Raup and Crick analyses suggested that total bacterial soil communities were subject deterministic selection becoming significantly similar as the experiment progressed, but this was inhibited by the highest concentration of additional Pb and Zn. Actinobacterial communities showed a similar response but were less affected by elevated Pb and Zn concentrations. These data indicate that the diversity of the actinobacterial community was not negatively affected by additional heavy metal stress in contrast to total bacterial community diversity.     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

Soil microbial biomass,crop yields,and bacterial community structure as affected by long-term fertilizer treatments under wheat-rice cropping

Soil microbial biomass carbon (SMBC) and nitrogen (SMBN), soil microbial community structure, and crop yields were studied in a long-term (1982–2004) fertilization experiment carried out in Suining, Sichuan province of PR China. Eight treatments included three chemical fertilizer (CF) treatments (N, NP, NPK), three CF + farmyard manure (M) treatments (NM, NPM, NPKM), M alone and no fertilizer (CK) as control. The results showed that the soil microbial biomass was higher in soil treated with CFM than in soil treated with CF alone, and that NPKM gave the highest rice and wheat yields. The SMBC and SMBN were higher after rice than those after wheat cropping. SMBC correlated closely with soil organic matter. Average yields of wheat and rice for 22 years were higher and more stable in the fertilized plots than in control plots. Bacterial community structure was analyzed by PCR-DGGE targeting eubacterial 16S rRNA genes. A higher diversity of the soil bacterial community was found in soil amended with CFM than in other fertilizer treatments. Some specific band emerged in the soil amended with M. The highest diversity of bacterial communities was found in the NPKM treated soil. The bacterial community structures differed in rice and wheat plots. Sequencing of PCR products separated in DGGE showed that some of the common and dominant bands were closely related to Aquicella lusitana and to Acidobacteria. This study demonstrated that mixed application of N, P, and K with additional M amendment increased soil microbial biomass, diversified the bacterial communities and maintained the crop production in the Calcareous Purplish Paddy soil.     

阔叶林改种毛竹(Phyllostachys pubescens)后土壤固氮细菌 nifH 基因多样性的变化

【目的】毛竹是喜氮植物,土壤氮素水平对毛竹生长至关重要。生物固氮是土壤氮素的重要来源,因此,探索阔叶林改种毛竹后土壤固氮细菌和土壤氮素的变化具有重要意义。【方法】选择立地条件相近的毛竹林(100多年前由阔叶林改种而来)和阔叶林,每种林地在东北坡向位置随机选择4个10 m×10 m标准样地,每个标准样地选取5个采样点,分层采集0—20 cm(表层)和20—40 cm(次表层)土壤样品,分析了土壤pH、有机碳、碱解氮、有效磷、速效钾和含水量等常规理化性质; 采用引物对AQER和PolF,以土壤总DNA为模板扩增了固氮细菌功能基因(nifH )片段,应用变性梯度凝胶电泳(DGGE)和实时荧光定量PCR(Real-time PCR),分析了固氮细菌群落结构、多样性以及丰度(nifH 基因拷贝数)变化; 通过基因克隆测序对土壤固氮细菌进行初步鉴定。【结果】阔叶林改种毛竹后土壤pH显著(P0.05)提高; 毛竹林土壤的含水量、碱解氮以及表层土壤的速效钾显著高于(P0.05)同层的阔叶林土壤,而有效磷则显著(P0.05)低于同层的阔叶林土壤。总体来说,阔叶林改种毛竹后土壤养分含量明显提高; 阔叶林土壤固氮细菌DGGE条带数以及多样性指数(Shannon-Wiener index)都高于毛竹林; 基于DGGE条带信息的聚类分析和主成分分析(PCA)结果表明,阔叶林和毛竹林区分为2个类群,而同一林分的不同土层之间差异较小; 实时荧光定量PCR结果显示,毛竹林土壤的固氮细菌 nifH 基因丰度显著(P0.05)高于阔叶林土壤; 通过克隆测序,14个阳性克隆分别属于2个不同的菌属,其中13个均为Bradyrhizobium,1个为Azohydromonas lata,条带序列与已知序列的相似度为93%~98%。【结论】阔叶林改种毛竹后土壤固氮细菌的种类减少,而功能基因丰度却明显增加; 土壤氮素水平明显提高,这可能是土壤固氮能力增强的结果。     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

Effects of Endosulfan on the Populations of Cultivable Microorganisms and the Diversity of Bacterial Community Structure in Brunisolic Soil

Endosulfan, an organochlorine pesticide, has been applied ubiquitously worldwide. However, endosulfan has been identified as a type of persistent organic pollutants (POPs), and its ecotoxicity has drawn attentions from scientists. The present study was implemented to examine the effects of endosulfan on the diversity and structure of soil microorganism communities. A control treatment and three concentrations (0.1, 1.0, and 10.0 mg/kg) were set up in laboratory experiments and sampled on days 7, 14, 21, and 28. The results revealed that the populations of bacteria and actinomycetes decreased significantly after 1.0 and 10.0 mg/kg treatments and that the soil microbial biomass carbon (MBC) was increased by endosulfan compared with the control. Terminal restriction fragment length polymorphism (T-RFLP) results revealed that the soil bacterial diversity was decreased by endosulfan and that the soil microbial community structure became unstable after endosulfan application. Moreover, the results of a 16S rRNA clone library revealed that the phyla Proteobacteria, Actinobacteria, Bacteroidetes, Spirochaetes, and Firmicutes showed an obvious advantage and closely relative. In conclusion, the results of the present study indicated that 0.1–10.0 mg/kg endosulfan showed obvious influences on the diversity and structure of the soil microbial community.     

纳米TiO2对黄瓜叶围细菌群落的影响

利用可培养和变性梯度凝胶电泳(DGGE)方法,对经不同浓度TiO2处理黄瓜(Cucumis sativus)叶围细菌群落结构的变化进行了分析。研究发现,TiO2处理后黄瓜叶围可培养细菌数量较对照显著降低。随着TiO2浓度从0.02 mg/mL提高至20 mg/mL,黄瓜叶围可培养细菌数量从1.8×107 cfu/g降低至3.1×106 cfu/g。DGGE指纹图谱分析表明,当TiO2的喷雾浓度超过0.02 mg/mL时,黄瓜叶围细菌的多样性显著降低。对DGGE条带克隆测序结果显示,在黄瓜叶围至少存在7种不同属的细菌,其中只有一种叶围细菌不受TiO2喷雾浓度影响。     

Clear-cutting affects the ammonia-oxidising community differently in limed and non-limed coniferous forest soils

The effects of clear-cutting on the ammonia-oxidising bacterial community were studied in the soil of limed and non-limed spruce forest plots located in the central part of southern Sweden. The communities were studied using denaturing gradient gel electrophoresis (DGGE) profiling after polymerase chain reaction (PCR) amplification from total DNA with primers reported to be specific for -subgroup ammonia-oxidising bacteria. The bands on the DGGE were sequenced and each unique sequence was interpreted as representing one ammonia-oxidising population. The relative abundance of each population was determined by measuring the fluorescence of the respective DGGE bands. In both limed and non-limed soil, the same two Nitrosospira populations were found, one belonging to cluster 2 (NScl2) and one to cluster 4 (NScl4). However, while NScl4 first appeared a year after the clear-cutting in the non-limed plot, it was present both before and after the cutting in the limed plot. Irrespective of previous liming, clear-cutting caused a shift in the ammonia-oxidiser community, from dominance by the NScl2 population to a community with approximately equal relative abundance of NScl2 and NScl4. In both plots the total size of the community increased after clear-cutting (based on increased DGGE band intensity), most likely due to increased NH₄⁺ availability, but the growth response was faster in the limed plot. Hence, the prior liming increased the responsiveness of the ammonia-oxidisers to the changes caused by cutting. This is the first study to report the effects of clear-cutting on the ammonia-oxidising community, and the results show a clear correlation between increased potential nitrification and a shift in the ammonia-oxidiser community.     

Amendment of degraded desert soil with wastewater debris containing immobilized Chlorella sorokiniana and Azospirillum brasilense significantly modifies soil bacterial community structure, diversity, and richness

The main goal of this study was to expand our knowledge of what happens to the soil bacterial community in an eroded desert soil when improvement of soil fertility is derived from the application of debris of tertiary wastewater treatment containing immobilized microalgae Chlorella sorokiniana and the plant growth-promoting bacterium (PGPB) Azospirillum brasilense. We hypothesized that an “improved” non-agricultural desert soil will exhibit substantial changes in the structure of the bacterial community in a relatively short time after amendment. To assess the effect of the amendments, microalgae and PGPB alone or combined, on the structure of the rhizosphere bacterial community, changes in species richness and bacterial diversity over time were based on sequence differences in the 16S rRNA gene, performed with PCR–denaturing gradient gel electrophoresis (DGGE) and then analyzed by similarity test and non-metric multidimensional scaling analysis. Root surface colonization and persistence in the rhizosphere of A. brasilense was monitored by fluorescent in situ hybridization and sequencing of DGGE bands. Application of waste debris significantly changed the rhizosphere bacterial population structure, whether comparisons were made over time, between inoculated and non-inoculated soil, and among different inoculated microorganisms. Species richness and diversity increased when the waste debris contained the microalgae–bacteria association and also over time. Even as its secondary role as an inoculant after wastewater treatment, A. brasilense colonized the root surface profusely and persisted within the rhizosphere bacterial community. This study demonstrated that small organic amendment to desert soil significantly changed soil bacterial community compared to the original soil and also 2 months after amendments were added.     

不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。